The prevalence of Chronic Kidney Disease (CKD) in the Veteran population is estimated to be a third higher than in the general population and the Veterans Health Administration currently cares for over 200,000 Veterans with moderate to severe kidney disease. The leading cause of death in patients with CKD is cardiovascular disease. Vascular calcification is highly prevalent in chronic kidney disease (CKD) and is a major cause of cardiovascular morbidity and mortality. Work done during our previous VA Merit Awards in vitro and in vivo in our CKD animal model, have demonstrated that vascular smooth muscle cells (VSMC) can de-differentiate to a more synthetic, osteo-chondrocytic cell with upregulation of RUNX2. This renders cells that can calcify in a manner similar to bone. Calcifying VSMC release matrix vesicles, small exosomes filled with calcium, phosphorus, protein and micro RNA (miRNA). Our bioinformatic analyses of miRNA arrays comparing VSMC and matrix vesicles have identified altered expression of multiple miRNA that regulate genes involved in VSMC phenotypic change/calcification, intracellular calcium (iCa) homeostasis, and oxidative stress pathways. Consistent with these findings, in vivo in our animal model of slowly progressive CKD there is also progressive increases in oxidative stress and iCa in VSMC. We hypothesize that Increased intracellular calcium (iCa) and oxidative stress are additive in inducing arterial calcification in CKD. To test this hypothesis in Aim 1, we propose to determine the relationship between increased intracellular calcium (iCa) and oxidative stress in calcifying VSMC from CKD versus NL animals. We will alter the iCa and determine the effect on oxidative stress, and conversely, decrease oxidative stress and determine if this alters iCa and if calcium channels/transporters are oxidized leading to altered function. In Aim 2, we will determine if the combination of decreased iCa and decreased oxidative stress alters CKD VSMC matrix vesicle protein content, miRNA profile, and modulation of the recipient VSMC osteogenic phenotype. In Aim 3, we will determine if the administration of the combination of agents to reduce both iCa and oxidative stress can prevent and/or treat arterial calcification in vivo in our rat model of progressive CKD. These studies will determine if the vicious cycle of increased iCa and increased oxidative stress accelerate vascular calcification in CKD, the mechanisms by which this happens, the role of matrix vesicle miRNA, and if dual blockade can prevent and/or stop arterial calcification that plaques patients with CKD. Our long term goal is to improve the cardiovascular health of Veterans with CKD.